Comparative Evaluation of the Effect of Two Platelet Concentrates (a-PRF and L-PRF) on the Cellular Activity of Pre-osteoblastic MG-63 Cell Line: An in vitro Study

Statement of the Problem: Currently, the reconstruction of bone defects with new platelet concentrates is considered a significant challenge in periodontics. Purpose: This study aimed to evaluate advanced- platelet rich fibrin (A-PRF) and leukocyte- and platelet rich fibrin’s (L-PRF) effects on the proliferation and differentiation of MG-63 cells. Materials and Method: In this in vitro study, blood samples of five healthy non-smoking volunteers were collected and immediately centrifuged according to the two protocols of Choukroun and Ghanaati, without adding any anticoagulants, to prepare L-PRF and A-PRF. After freezing the clots for one hour, they were crushed and centrifuged once more. After culturing MG-63 cells, the effects of 20%, 10%, 1%, and 0.5% concentrations of A-PRF and L-PRF extracts on cell proliferation and mineralization were evaluated by methyl thiazolyl tetrazolium (MTT) assay and Alizarin Red staining, respectively. Results: Generally, survival and proliferation in the L-PRF group at both time intervals were higher than the A-PRF group and increased with increasing the extract concentration. However, in the A-PRF group, there were no significant differences between the different concentrations, and only the number of cells increased over time. After three days, in the study on mineralization, nodule formation was observed only in the positive control group (osteogenic). In seven days, mineralized nodules were formed in all groups with different concentrations of A-PRF, but not in any of the L-PRF groups. Conclusion: According to the results, L-PRF increased proliferation, and A-PRF exerted a positive effect on the differentiation of MG-63 cells.


Introduction
Reconstruction of bone defects due to trauma, infection, tumor, and congenital lesions is a critical clinical problem. Despite the availability of allografts and xenografts, the use of these bone substitutes is limited due to the risk of host immune response and the possibility of pathogen transmissions. Fortunately, new advances in tissue engineering techniques have provided promising alternatives for bone defect reconstruction [1]. Tissue engineering requires a cellular scaffold that provides the physiological state where bone cells can migrate, proliferate, and differentiate to form new bone [2]. Accordingly, the role of growth factors in wound healing and periodontal tissue regeneration is critical [3].
The second generation of platelet concentrates, called Choukron's platelet rich fibrin (PRF) or leuko-cyte-and platelet rich fibrin (L-PRF), was introduced in 2006 [4]. Due to the lack of any anticoagulants and biochemicals in PRF preparation, physiological polymerization of fibrin provides a network for cellular migration and proliferation, and thus for wound healing.
Activation and degranulation of platelets and leukocytes during centrifugation results in release of cytokines and growth factors from L-PRF significantly during fibrin matrix remodeling. In addition to inflammation regulation [5][6], L-PRF serves as a reservoir of the most significant growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGFβ), and insulin-like growth factor (IGF) [3,[5][6][7].
These factors accelerate the cellular cycle, activate angiogenesis, speed up scar tissue formation and wound closure, and cause healing without infection [8]. Furthermore, using L-PRF does not require exogenous scaffolds as a carrier for cells [1]. The PRF provides an appropriate scaffold that maintains cytokines and facilitates osteogenic differentiation [9][10]. L-PRF exudates also enhance the proliferation and osteogenic differentiation of periodontal ligament cells in vitro [11]. Wang et al. [12] showed that the application of L-PRF with mesenchymal stem cell sheets resulted in the healing of rabbit's calvarial bone lesions in critical size.
In 2014, Ghanaati et al. [13] introduced a new product called advanced-platelet rich fibrin (A-PRF). They found that a change in centrifugation force (reducing revolutions per minute and increasing time) in the preparation of L-PRF resulted in a better distribution pattern of monocytes and B and T cells in the whole membrane.
The A-PRF releases a large amount of TGFβ, IGF, PDGF, and epidermal growth factor in 10 days, and also exhibits high levels of and collagen type I mRNA [14].
A-PRF has a higher content of PDGF, TGFβ and vascular endothelial growth factor (VEGF) than other platelet concentrates like platelet-rich plasma, concentrated growth factor, and plasma rich in growth factor and significantly induces in vitro proliferation in human periosteal cells [15]. It is also indicated that the A-PRF membrane has the highest ability to release TGFβ, PDGF, VEGF, and epidermal growth factor for 10 days compared to the L-PRF [16].
Considering the uniform, continuous, and long-term release pattern of growth factors [17], the A-PRF can be used as an appropriate membrane to improve bone mar-row proliferation and differentiation. Moreover, A-PRF alone or in combination with freeze-dried bone allograft compared to freeze-dried bone allograft alone was a more suitable biomaterial for ridge preservation; the amount of live bone volume and mineral density in samples of patients treated by A-PRF was significantly higher, and ridge height reduction was low [18]. However, contradictory results have been reported in another study [19].
Increasing the clinical use of platelet membranes (like L-PRF and A-PRF) for improving bone remodeling around teeth and implants as well as their easy application would justify the need for further studies on these platelet products. Due to the lack of laboratory information about their effects on osteoblasts, this in vitro study aimed to evaluate the influence of different concentrations of A-PRF and L-PRF extracts on the cellular activity of pre-osteoblastic MG-63 cells for the first time.

Materials and Method
This randomized controlled single-blind in vitro trial was conducted to evaluate the effect of A-PRF and L-PRF extract on proliferation and mineralization of MG-63 cells. The study population was pre-osteoblastic MG-63 cells that were cultured in cell culture plates with an initial density of 50,000 cells.
The cells were passaged every three days, and when cell confluency reached approximately 80%, they were detached from the flasks by trypsin-EDTA and counted under an optical microscope using trypan-blue dye and a Neubauer plate. Then the cells were divided into two groups. A group with a density of 3000/well was transferred to two 96-well plates (each containing 200 µL) to assay cell proliferation at 24 and 72 hours. The other group was transferred to two 24-well plates with a density of 30,000/well for three days and 10,000/well for seven days for histological evaluation of calcified nodule formation using Alizarin red staining. The cells were incubated for 24 hours to achieve monolayer cell growth and then washed with phosphate-buffered saline solution (Gibco, USA). The previous culture medium was replaced with a 1% FBS medium for 24 hours (starving stage) [20]. All the steps were performed under sterile conditions and a biological hood (Laminar Flow, Class II, Arster, Iran). After explaining the study procedure and obtaining informed consent forms, 50 samples of 9 mL blood from the antecubital vein of these individuals (under sterile condition) were placed in 10-mL dry glass-coated plastic tubes (Blood collecting tube, Intra-spin, Intralock, USA), which are specialized for PRF preparation.
The tubes were randomly (coin toss) divided into L-PRF and A-PRF after collection. The tubes were centrifuged according to the Choukroun [4] (2700 revolutions per minute, 12 minutes) and Ghanaati [13] protocols (1500 revolutions per minute, 14 minutes) for L-PRF and A-PRF preparation, respectively, without adding any anticoagulant. Then they were transferred under a biosafety cabinet. The fibrin clots of L-PRF and A-PRF were held with a forceps, cut off by scissors from above the red corpuscles layer, and transferred to the PRF box.
It was noted that the buffy coat part did not separate from the fibrin clot.
The clots were converted to membranes in the PRF box after the serum disappeared. Then, the L-PRF and A-PRF membranes were separately frozen within the nitrogen tank for one hour at -80°C. After one hour, the frozen membranes were cut to small pieces by surgical blades and sterile scissors, and were centrifuged again (400g, 10min). The upper fluid of tubes (PRF extract) was collected, and 20%, 10%, 1%, and 0.5% concentrations of A-PRF and L-PRF extracts were prepared in Dulbecco's modified eagle medium containing 1% FBS [15].

Viability and cell proliferation
MTT assay was used to evaluate the effect of different concentrations of L-PRF, A-PRF extracts (20%, 10%, 1%, and 0.5%) on cell viability, and proliferation compared to describe positive and negative control groups [20]. After the cells starved in 1% FBS for 24 h, the triplicated test was performed by one individual at specified times. The cells were exposed to different concen-

Examination of mineralization (Alizarin Red staining)
Differentiation evaluation was performed by observing the formation of mineralized nodules with Alizarin Red staining. The cells were placed in two 24-well plates and exposed to 20%, 10%, 1%, and 0.5% PRF extracts and negative and positive control groups (culture medium containing 1% FBS and osteogenic medium, respectively) for three and seven days. The test was performed in duplicate. After evacuating the medium, the cells were washed with phosphate-buffered saline solution and fixed with cold ethanol for an hour, and then washed twice with water for five minutes each time.
After washing, 2% Alizarin Red solution was placed on the cells for 30 minutes at room temperature. The colored solution was then removed, and the cells were rinsed with water four times for five minutes each time.
The plates were investigated carefully under an inverted optical microscope at 20× and 40× magnifications to evaluate the cells' morphology and detect mineralized nodules colored red to orange. The results of this staining examination were qualitatively reviewed and reported.

Viability and cell proliferation evaluation by MTT assay
MTT assay was used to evaluate the effect of different concentrations (20%, 10%, 1%, and 0.5) of L-PRF and A-PRF extract on cell viability and proliferation. In this test, the cell viability in the negative control group (containing 1% FBS medium) was considered 100% and the average light absorption of each experimental group was expressed as a percentage compared to the control group (Table 1).
In both groups, the cell proliferation at 72 hours was greater than 24 hours. Compared to the control group, the average percentage of cells after 72 hours (392±8) was more than 24 hours (114±8). It means that the influence of time on the amount of proliferation in 72 hours was significantly higher than 24 hours (p< 0.001).
In each experimental group, except for the control groups containing water and 1% FBS medium, the increase in the number of cells in 72 hours was significantly higher than 24 hours (p< 0.001). However, the effect of A-PRF and L-PRF on MG-63 cells' proliferation in 24 hours was not significant (p> 0.05). Two-bytwo comparison of different concentrations of the A-PRF and L-PRF extracts, showed no significant differences between the two extracts in the stimulation of cell   Table 2).

Examination of mineralized nodules formation (Alizarin Red staining)
A qualitative study of mineralized nodule formation in

Discussion
Our results showed that only higher concentrations of L-PRF (10% and 20%) significantly increased the proliferation of MG-63 cells compared to other groups. In addition, seven days after exposure, only A-PRF (even at low concentrations) stimulated the production of calcium nodules. According to the results, these products might be used to repair bone defects.
The size of PRF clots created in this study varied in different individuals and even in several samples of one.
Therefore, for standard comparison, it seems that using equal numbers of tubes from A-PRF and L-PRF and obtaining similar concentrations of extracts provide more similar conditions than using full-size clots with different sizes. In the present study, extracts were used instead of PRF membranes (by centrifuging for 10 minutes at 400 g force after shredding the membranes), which minimized the interfering factors useful in forming the membranes.
In the present study, the MTT test showed that the rate of cell proliferation in the A-PRF group, despite the similar concentration, was less than half of that in the L-PRF group 72 h after exposure to extracts. This would possibly reflect the different characteristics of these two  In another study, Dohan et al. [24] showed that the effect of L-PRF on osteoblasts was always dosedependent compared to other cells and consistently stimulated cell proliferation up to 28 days. This enhancing effect of L-PRF on proliferation can be justified by releasing PDGF and TGFβ, which have been shown to have mitogenic effects on cells [25].
In the present study, cell proliferation in the 20% L-PRF extract group was more than that in the 10% L-PRF group in 72 hours, but the differences were not statistically significant, which might be due to its dosedependent effect on cell proliferation up to a certain dose of extracts. This has also been observed previously with other forms of platelet extracts. Graziani et al. [26] showed that optimum platelet-rich plasma's platelet extract concentration affecting osteoblasts proliferation was 2.5 times over its blood concentration. Further studies should be carried out using higher L-PRF concentrations to obtain the maximum concentration of MG-63 proliferative platelet extract.
In the present study, the proliferation rate of MG-63 cells in the L-PRF group was significantly higher than that in the negative control group (10% FBS). Hence, it might be prudent to use lower concentrations of L-PRF extract instead of FBS in human cell proliferation studies, which results in eliminating the risk of infection and immune responses [27]. However, the possibility of its undesirable effects on the growth of cells should be con-sidered.
In addition to evaluating cell proliferation, we stud- cells might begin to differentiate after reaching maxim-um confluency [35].
Osteoblasts require at least 14 days to mineralize, and the use of other cell differentiation assays, such as the evaluation of alkaline phosphatase activity and the presence of type I collagen [24], will be helpful in a shorter time. In addition, despite the lower expression of bone sialoprotein and osteocalcin in MG-63 cells [33], it might be possible to resolve this problem by examining these osteogenic markers as well as osteopontin, which is expressed at a higher rate in the late stages of osteoblast differentiation [28].
In other studies, evaluation of mineralization in the dental follicle and periodontal ligament cells [22] and dental pulp stem cells [29] [36].
Macrophages are also a source of chemotactic agents essential for the stimulation of angiogenesis [31] and have a special role in the secretion of growth factors such as TGFβ, PDGF, and VEGF [36]. These growth factors and hormones regulate bone regeneration by affecting cell differentiation and cell growth regulation [28].
Moreover, Chen et al. [37], in a systematic review of randomized controlled trials suggests that platelet-rich products enhance wound healing.
Bone formation is strongly controlled by bone and endothelial cell interactions in the healing region. In addition, biological factors like TGFβ, BMPs, FGF, PDGF, and IGF can potentially be used in bone regeneration [28]. The synergistic effect of BMP-2 and VEGF by stimulating osteogenesis and angiogenesis [38] and the importance of BMP-2 in osteogenesis are well known. The positive effects of BMP-2, BMP-7, IGF-1, and PDGF on the regeneration of periodontal lesions have been reported, and RUNX2 and BMP have been identified as the most potent regulators of osteoblastic differentiation in mesenchymal stem cells [28].
The role of PRF in the overexpression of bone sialoprotein, osteocalcin, alkaline phosphatase, and RUNX2 genes has been demonstrated [39]. Ghanaati et al. [13] in a comparison of these two platelet products has shown A-PRF to have a looser structure with more spaces between the collagen fibers and more cells in its fibrin clot than L-PRF. Besides, the existing cells are more evenly distributed throughout the fibrin clot. However, L-PRF cells are more concentrated in the area near the red blood cells. A-PRF releases higher growth factors than L-PRF, which is attributed to its higher leukocyte content. Dohan et al.'s research results [19] contradict this outcome. In their report, the slow release of PDGF-AB, TGFβ1, and VEGF from the L-PRF membrane at all time intervals was significantly (more than twice) higher than that of A-PRF. BMP-2 was not detected in the A-PRF membrane and was slowly released from the L-PRF for seven days. As observed in the present study, the L-PRF membrane and clot in all the samples were significantly larger and stronger than the A-PRF samples.

Conflict of Interest
Authors declare that there are no conflicts of interest in this study.